Methods for the preparation of plants with changed sucrose concentration by transformation with a sucrose phosphate synthase nucleic acid

This application is a divisional of application Ser. No. 08/778,656 filed Jan. 3, 1997, now U.S. Pat. No. 5,976,869, which issued on Nov. 2, 1999, which is a divisional of application Ser. No. 08/356,354 filed Dec. 20, 1994, now U.S. Pat. No. 5,767,365, which issued on Jun. 16, 1998, which is a continuation of PCT/EP93/01605 filed Jun. 22, 1993. The disclosure of these applications are hereby incorporated by reference into the present application.

The present invention relates to DNA sequences and plasmids, containing these DNA sequences, which by integration into a plant genome, cause the activity of the sucrose-phosphate-synthase (SPS) of the plant to be changed and thus affect the sugar metabolism of the plant. The invention further relates to transgenic plants, in which through introduction of the DNA sequences, changes in the activity of the sucrose-phosphate-synthase are produced.

Sucrose is of central importance for the plant and serves many functions. For the long distance transport of photoassimilates and/or energy between various organs in plants, sucrose is almost exclusively used. The sucrose which is transported in a specific heterotrophic organ determines the growth and the development of this organ. Thus it is known, e.g. from EP 442 592, that transgenic plants, in which the transport of the sucrose away from the exporting leaves is inhibited by expression of an apoplastic invertase, shows a strong reduction in the growth of e.g. roots or tubers in the case of potato plants. For tobacco plants, the principal importance of sucrose for the long distance transport of energy carriers within the plant is described in von Schaewen et al, 1990, EMBO J 9: 3033-3044.

While it has been clearly shown that a reduction of the amount of sucrose imported in the heterotrophic organs, such as tubers and seeds, leads to loss of yield, it is not known whether an increase in the amount of sucrose in the photosynthetically active parts of the plant, mainly the leaves, leads to a better supply of heterotrophic organs and thus to an increase in yield.

A second central role for sucrose and/or the hexoses, glucose and fructose which are derived from sucrose, is in the protection of plants against frost damage at low temperatures. Frost damage is one of the main limiting factors in agricultural productivity in the northern hemisphere. Temperatures below freezing lead to the formation of ice crystals. Since the growing ice crystals consist of pure water, water is abstracted from the cells as the temperature falls.

This dehydration has at least two potential damaging results:

a) all dissolved substances within a cell are strongly concentrated and the cell contracts following the loss of water. Highly concentrated salts and organic acids lead to membrane damage;

b) with rehydration from dew, the previously contacted cells reexpand. The cell membrane also expands again. The volume expansion puts a heavy mechanical load on the membrane.

It is thus clear that a freezing/dew cycle can lead to severe membrane damage of the cells and thus to damage to the plant.

It thus appears worth trying to hinder the freezing. One possible strategy is to increase the formation of osmotically active substances in the cytosol of plant cells. This should lead to a lowering of the freezing point. Osmotically active substances include sucrose and/or the two hexoses which are derived from sucrose.

The increased formation of sucrose and/or the two hexoses at low temperatures is desirable in the growing plant. Another situation can exist in the harvested parts of a plant, especially in storage. For example, in potato tubers that are stored at 4-8° C., hexoses (glucose) accumulate. It would appear to be sensible, to see this as the answer to a lowering of the temperature (“cold-sweetening”).

The accumulation of sucrose and glucose has in the case of potato tubers economically undesirable results. Increased amounts of reducing sugars, such as glucose, in potatoes which are fried when preparing crisps, chips and the like, leads to an undesirable browning due to the Maillard reaction. Such products with a dark brown color are not generally acceptable to the consumer. Further the cooking strength is strongly dependent on the content of starch and/or its breakdown products which are important in determining the quality characteristics of the potato.

In relation to the economic aspects, sucrose thus possesses three especially important functions:

1 as the transport form for the distant transport of photoassimilates,

2 as an osmotically active substance with the desirable activity of lowering the freezing point in intact, growing plants, and

3 in the undesirable formation of reducing sugars in stored harvested parts of a plant, e.g. the potato tubers, as a result of low temperatures.

The biosynthesis pathways for the formation of sucrose, either from the primary photosynthesis products (in the leaf) or by breakdown of starch (in the storage organs e.g. of potatoes), are known. An enzyme in sucrose metabolism is sucrose-phosphate-synthase (SPS). It forms sucrose-6-phosphate from UDP-glucose and fructose-6-phosphate, which in a second step is converted to sucrose.

The isolation of SPS from maize and the cloning of cDNA from mRNA from maize tissue is known (EP 466 995). In this application, processes for the purification of a protein such as by centrifuging or homogenates, differential precipitation and chromatography are described. A 300 times enrichment of SPS from plant tissue has been described by Salerno and Pontis (Planta 142: 41-48, 1978).

In view of the significance of SPS for carbohydrate metabolism, it is questionable whether plants can tolerate a reduction in SPS activity in all or in certain organs. It is especially not known whether it is possible to produce transgenic plants with a reduced SPS activity. Also the use of SPS for the modification of the functions of sucrose for lowering the freezing point in intact plants and for the formation of reducing sugars in harvested parts is not known.

For the preparation of plants with reduced SPS activity, i.e. plants with changed sucrose concentration, it is necessary to make available an SPS coding region of such plant species, for which processes are described, whereby transgenic plants can be grown in large numbers. In as much as a reduction of SPS activity can be achieved, by selection from a large amount, the possibility exists of obtaining plants with such a phenotype. Further organ specific promoters for gene expression should exist for the plant species, by which the possibility of an organ specific reduction of the SPS activity could be investigated.

A species which fulfils the stated requirements is Solanum tuberosum . The genetic modification of Solanum tuberosum by gene transfer using Agrobacteria is well described (Fraley et al., 1985, Crit Rev Plant Sci 4: 1-46). Promoters for leaf specific (Stockhaus et al., 1989, Plant Cell 1: 805-813), tuber specific (EP 375 092) and wound inducing (EP 375 091) gene expression are known.

The present invention now provides DNA sequences with which changes of SPS activity are actually and demonstrably possible and with which the sucrose concentration in the plant can be modified. It is concerned with sequences which include the coding region of sucrose-phosphate-synthase (SPS) from Solanum tuberosum.

These DNA sequences can be introduced in plasmids and thereby combined with steering elements for expression in eukaryotic cells. Such steering elements are, on the one hand, transcription promoters and, on the other hand, transcription terminators.

Each plasmid comprises:

a) a suitable promoter that ensures that the coding sequence is read off at the suitable time point and/or in a specified development stage in the transgenic plants or in specified tissues of transgenic plants,

b) at least one coding sequence, that

i) is coupled to the promoter so that RNA can be translated into protein, whereby the protein demonstrates enzymatic activity, that leads to a modification of the sucrose concentration in the plant, or

ii) is coupled to the promoter so that the non-coding strand is read off, which leads to the formation of a so-called “anti-sense” RNA, which suppresses the formation of the coding protein of an endogenous gene in the plant which is involved in the sucrose biosynthesis, and

c) a non-coding termination sequence that contains the signals for the termination and polyadenylation of the transcript.

The present invention further provides plasmids in which include DNA sequences which change the SPS activity in the plant.

The coding sequences named under b) include the SPS sequences with the following nucleotide sequences:

All sequences are cDNA sequences and stem from a cDNA library of leaf tissue. The expression gene is the same in various plant tissues. As a promoter, there can generally be used any promoter which is active in plants. The promoter should ensure that the foreign gene is expressed in the plant. The promoter can be chosen so that the expression occurs only in specified tissues, at a determined time point in the plant's development or at a time point determined by outside influences. The promoter can be homologous or heterologous to the plant. Suitable promoters are e.g. the promoter of the 355 RNA of the cauliflower mosaic virus, the patatin promoter B33 (Rocha-Sosa et al. (1989) EMBO J 8: 23-29) or a promoter that ensures expression only in photosynthetically active tissues. Other promoters can be used which ensure expression only in specified organs, such as the root, tuber, seed, stem or specified cell types such as mesophyllic, epidermal or transport cells. For hindering cold sweetening, suitable promoters are those which ensure an activation of the transcription is stored in harvested parts of the plants. For this, there can be considered cold induced promoters or such promoters that become active during the transition of the tuber from the phase where it stores material to the phase where it gives up material.

FIG. 1 provides the structure of the 35S-anti-pot-SPS gene;

FIG. 2 provides the structure of the B33-anti-pot-SPS gene;

FIG. 3 provides an analysis of plants transformed with a plasmid which included the 35S-anti-pot-SPS gene; and

FIG. 4 is an analysis of plants transformed with a plasmid which included the B33-anti-pot-SPS gene.

The coding sequence contains the information for the formation of an mRNA for the sucrose-phosphate-synthase (SPS) or for the formation of an anti-sense RNA for the SPS. Whether the translatable MRNA or an anti-sense RNA is formed depends on the orientation of the coding sequence in relation to the promoter. If the 3′ end of the coding sequence is fused to the 3′ end of the promoter, an anti-sense RNA results, while the fusion of the 5′ end of the coding sequence to the 3′ end of the promoter promoter, a translatable RNA results. This latter leads to an increase of the SPS activity in the cell, while the first leads to a reduction of the SPS activity in the cell. Such a reduction of SPS activity is especially significant in view of the undesirable formation of sucrose and/or reducing sugars as a result of cold storage of harvested organs.

The coding sequence for SPS can be one of the three described above or one that is derived by modifications of the sequences described above. A derivation can be carried out, e.g. by current methods of mutagenesis and/or recombination. In particular, changes of SPS sequences that lead to a neutralisation of the plant's own regulation mechanism are contemplated.

The DNA sequences of the invention can also be used for the preparation of derivatives whose gene products are not subjected to the plant's own activity regulation during a phosphorylation reaction.

Further, the sequences can also be used for the preparation of derivatives by targeted and non-targeted mutagenesis.

The invention relates further to derivatives of the DNA sequences of the invention that are-obtained by exchange of single bases or by deletion or insertion of base sequences and which code for proteins with a comparable activity to sucrose-phosphate-synthase.

The 5′ untranslated area of the sequence Seq. ID No 1, is not part of SPS, but is shown as a cloning artefact. The methionine start codon of the coding region lies in a region in which there is no homology of the amino acid sequence to the other SPS sequences. Since this sequence does not also fully coincide in the homologous region with one of the other sequences, it is recognisable that the sequence Seq. ID No 1 is not a derivative of the sequences Seq. ID No 3 and Seq. ID No 5.

The termination sequence provides the correct finishing of the transcription and the attachment of a polyadenyl group to the RNA. This polyadenyl group has an important function in the stabilisation of RNA molecules in the cells. With suitable plasmids which contain the DNA sequences of the invention, plants can be transformed with the object of raising and/or reducing the SPS activity and/or modifying the sucrose concentration.

Plasmids that can be used include e.g. p35S-anti-pot-SPS (DSM 7125) and pB33-anti-pot-SPS (DSM 7124). With the gene 35S-anti-pot-SPS, located on the plasmid p355-anti-pot-SPS, the concentration of the mRNA for the SPS protein and the enzymatic activity, for example, can be reduced. With the gene B335-anti-pot-SPS, located on the plasmid pB33-anti-pot-SPS, the concentration of the mRNA for the SPS protein and the enzymatic activity, specifically for potato tubers for example, can be reduced. In a similar way to the SPS sequence (Seq. ID No. 1) located on this plasmid, other SPS sequences, e.g. the sequences Seq. ID No. 3 and Seq. ID No. 5 also be cloned in suitable vectors and for the same purpose.

In the plant, the SPS is subjected to an activity control by phosphorylation. This allows the plant to regulate the activity of the enzyme within a fixed frame independent of the amount of the SPS protein. If one of the changes occurring outside the activity of the SPS is to achieved, it is necessary to evade the plant's own regulation mechanism. Therefore changing the phosphorylation possibilities is an important target for influencing the SPS activity and thus the sucrose content of the plant.

It is not known in which position in the SPS protein target directed changes of the coding regions can be achieved which serve the purpose of introducing in the plant SPS activity which is not subject to any of the plant's own controls.

The DNA sequence described here, which contains the coding region for SPS from Solanum tuberosum , allows the identification of the sites of protein phosphorylation of the SPS. By using standard methods (Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular Cloning: A laboratory Manual, 2nd. Edn., Cold Spring Harbor Laboratory Press, NY, USA), a localisation of the phosphorylation positions of SPS is possible using the DNA sequences of the invention. These being known, by use of the plasmids with the SPS sequence, a target directed mutagenesis (Sambrook et al, 1989) of the coding region of SPS and/or a non-target directed mutagenesis (Sambrook et al, 1989) and subsequent probing of the desired mutations of the coding region of the SPS can be undertaken. Derivatives of the coding region can be prepared with the help of this plasmid, whose derived proteins are not subjected to the plants own regulation mechanisms.

Since the SPS enzyme is regulated by phosphorylation in all tested species, except maize, one can refer to sequence comparisons to identify possible phosphorylation sites. The criterium for this is that a serine residue appears in an acidic medium in the regulated SPS protein, but not however with maize. There are 12 such serine residues in the sequences, Seq. ID No. 3 and Seq ID No. 5. In the sequence Seq ID No. 1, the first of the 12 serine residues is missing, since the coding region begins just later. The sequence, Seq. ID No. 1 is thus especially suitable for the production of SPS activity in plants that is not liable to endogenous activity regulation.

For the introduction of the SPS sequence in higher plants, a large number of cloning vectors are available which contain a replication signal for E. coli and a marker, which allows for the selection of the transformed cells. Examples of vectors are pBR 322, pUC-series, M13 mp-series, PACYC 184; EMBL 3 etc. According to the introduction method of the desired gene in the plant, other DNA sequences may be suitable. Should the Ti- or Ri-plasmid be used, e.g. for the transformation of the plant cell, then at least the right boundary, often however both the right and left boundary of the Ti- and Ri-Plasmid T-DNA, is attached, as a flanking region, to the gene being introduced. The use of T-DNA for the transformation of plants cells has been intensively researched and is well described in EP 120 516; Hoekama, In: The Binary Plant Vector System, Offset-drukkerij Kanters B. V. Alblasserdam, (1985), Chapter V; Fraley, et al., Crit. Rev. Plant Sci., 4:1-46 and An et al. (1985) EMBO J. 4: 277-287. Once the introduced DNA is integrated in the genome, it is generally stable there and remains also in the offspring of the original transformed cells. It normally contains a selection marker, which induces resistance in the transformed plant cells against a biocide or antibiotic such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin etc. The individual marker employed should therefore allow the selection of transformed cells from cells which lack the introduced DNA.

For the introduction of DNA into a plant, besides transformation using Agrobacteria, there are many other techniques available. These techniques include the fusion of protoplasts, microinjection of DNA and electroporation, as well as ballistic methods and virus infection. From the transformed plant material, whole plants can be regenerated in a suitable medium which contains antibiotics or biocides for selection. The resulting plants can then betested for the presence of introduced DNA. No special demands are placed on the plasmids in injection and electroporation. Simple plasmids, such as e.g. pUC-derivatives can be used. Should however whole plants be regenerated from such transformed cells the presence of a selectable marker gene is necessary. The transformed cells grow within the plants in the usual manner (see also McCormick et al.(1986) Plant Cell Reports 5: 81-84). These plants can be grown normally and crossed with plants, that possess the same transformed genes or different genes. The resulting hybrid individuals have the corresponding phenotypical properties.

Deposits

The following plasmids were deposited at the Deutschen Sammlung von Mikroorganismen (DSM) in Braunschweig, Germany on the 12.06.1992 (deposit number):

Plasmid p35S-anti-pot-SPS (DSM 7125) Plasmid pB33-anti-pot-SPS (DSM 7124)

FIG. 1 : Structure of the 35S-anti-pot-SPS gene

A=Fragment A: CaMV 35S promoter, nt 6909-7437 (Franck et al.,1980, Cell 21: 285-294)

B=Fragment B: sucrose phosphate synthase, EcORV Fragment (nt 1 bis 2011), ca. 2000 bp, orientation: anti-sense

C=Fragment C: nt 11748-11939 of the T-DNA of the Ti-plasmid pTiACH5; Gielen et al., 1984, EMBO J 3: 835-846)

FIG. 2 : Structure of the B33-anti-pot-SPS gene

A=Fragment A: B33 promoter of the patatin gene from S. tuberosum , (Rocha-Sosa et al., 1989, EMBO J 8: 23-29), ca 530 bp

B=Fragment B: sucrose phosphate synthase (s. FIG. 2 ), EcoRV fragment (nt 2011 bis 1), ca. 2000 bp, orientation: anti-sense

C=Fragment C: nt 11748-11939 of T-DNA of the Ti-plasmid pTiACH5 (Gielen et al., 1984, EMBO J 3: 835-846)

FIG. 3 : shows the results of the transformation of transgenic potato plants.

Control=wild type plants

1-75=individual transgenic plants

FIG. 4 : shows the results of the transformation of potato plants

Control=wild type plants

3-20=individual transgenic plants

In order to understand the examples forming the basis of this invention all the processes necessary for these tests and which are known per se will first of all be listed:

1. Cloning Process

The vectors pUC 18/19 and M13mp10 series (Yanisch-Perron et al. (1985) Gene 33: 103-119), as well as the vector EMBL 3 (Frischauf et al. (1983) J Mol Biol 170: 827-842) were used for cloning.

For the plant transformations, the gene constructs were cloned in the binary vector BIN 19 (Bevan (1984) Nucl. Acids Res 12: 8711-8720)

2. Bacterial Strains

The E. coli strain BMH71-18 (Messing et al., Proc. Natl. Acad. Sci. USA (1977), 24, 6342-6346) or TB1 was used for the pUC and M13 mP vectors.

For the vector BIN19, only the E. coli strain TB1 was used. TB1 is a recombinant-negative, tetracycline-resistant derivative of strain JM101 (Yanisch-Perron et al., Gene (1985), 33, 103-119). The genotype of the TB1 strain is (Bart Barrel, personal communication): F′ (traD36, proAB, lacI, lacZΔM15), Δ (lac, pro), SupE, this, recA, Sr1::Tn10(TcR).

The transformation of the plasmids into the potato plants was carried out using Agrobacterium tumefaciens strain LBA4404 (Bevan, (1984), Nucl. Acids Res. 12, 8711-8720).

3. Transformation of Agrobacterium tumefaciens

In the case of BIN19 derivatives, the insertion of the DNA into the Agrobacterium was effected by direct transformation in accordance with the method of Holsters et al., (1978) (Mol Gene Genet 163: 181-187). The plasmid DNA of the transformed Agrobacterium was isolated in accordance with the method of Birnboim and Doly (1979) (Nucl Acids Res 7: 1513-1523) and was analysed by gel electrophoresis after suitable restriction cleavage.

4. Plant Transformation

Ten small leaves, wounded with a scalpel, of a sterile potato culture were placed in 10 ml of MS medium with 2% sucrose containing 30-50 μl of an Agrobacterium tumefaciens overnight culture grown under selection. After 3-5 minutes gentle shaking, the leaves were laid out on MS medium of 1.6% glucose, 2 mg/l of zeatin ribose, 0.02 mg/l of naphthylacetic acid, 0.02 mg/l of gibberellic acid, 500 mg/l of claforan, 50 mg/l of kanamycin and 0.8% bacto agar. After incubation for one week at 25° C. and 3000 lux, the claforan concentration in the medium was reduced by half.

5. SPS Activity Test

The SPS activity was determined according to the method of Siegel and Stitt (1990, Plant Science 66: 205-210) in a two stage analysis. To 180 μl of a solution of 50 mM HEPES/KOH (pH 7.4), 5 mM magnesium chloride, 5 mM fructose-6-phosphate, 25 mM glucose-6-phosphate and 6 mM uridine-5′-diphosphoglucose 20 μl of probe was added and incubated for 10 minutes at 25° C. It was heated for 3 minutes at 95°C., to complete the reaction. After centrifuging, the supernatant was spectroscopically analysed for the liberation of uridine-5′-diphosphate, whereby a pyruvate-kinase coupling enzyme reaction was used. Preparations without hexose phosphate, as well as the measurement of the recovery of added uridine-5′-diphosphate act as controls.

Solanum tuberosum

2

Met Arg Tyr Leu Lys Arg Ile Asn Met Lys Ile Trp Thr Ser Pro Asn

1 5 10 15

Ile Thr Asp Thr Ala Ile Ser Phe Ser Glu Met Leu Thr Pro Ile Ser

20 25 30

Thr Asp Gly Leu Met Thr Glu Met Gly Glu Ser Ser Gly Ala Tyr Ile

35 40 45

Ile Arg Ile Pro Phe Gly Pro Arg Glu Lys Tyr Ile Pro Lys Glu Gln

50 55 60

Leu Trp Pro Tyr Ile Pro Glu Phe Val Asp Gly Ala Leu Asn His Ile

65 70 75 80

Ile Gln Met Ser Lys Val Leu Gly Glu Gln Ile Gly Ser Gly Tyr Pro

85 90 95

Val Trp Pro Val Ala Ile His Gly His Tyr Ala Asp Ala Gly Asp Ser

100 105 110

Ala Ala Leu Leu Ser Gly Ala Leu Asn Val Pro Met Leu Phe Thr Gly

115 120 125

His Ser Leu Gly Arg Asp Lys Leu Glu Gln Leu Leu Arg Gln Gly Arg

130 135 140

Leu Ser Lys Asp Glu Ile Asn Ser Thr Tyr Lys Ile Met Arg Arg Ile

145 150 155 160

Glu Ala Glu Glu Leu Thr Leu Asp Ala Ser Glu Ile Val Ile Thr Ser

165 170 175

Thr Arg Gln Glu Ile Asp Glu Gln Trp Arg Leu Tyr Asp Gly Phe Asp

180 185 190

Pro Ile Leu Glu Arg Lys Leu Arg Ala Arg Ile Lys Arg Asn Val Ser

195 200 205

Cys Tyr Gly Arg Phe Met Pro Arg Met Ala Val Ile Pro Pro Gly Met

210 215 220

Glu Phe His His Ile Val Pro His Glu Gly Asp Met Asp Gly Glu Thr

225 230 235 240

Glu Gly Ser Glu Asp Gly Lys Thr Pro Asp Pro Pro Ile Trp Ala Glu

245 250 255

Ile Met Arg Phe Phe Ser Asn Pro Arg Lys Pro Met Ile Leu Ala Leu

260 265 270

Ala Arg Pro Asp Pro Lys Lys Asn Leu Thr Thr Leu Val Lys Ala Phe

275 280 285

Gly Glu Cys Arg Pro Leu Arg Glu Leu Ala Asn Leu Thr Leu Ile Met

290 295 300

Gly Asn Arg Asp Asn Ile Asp Glu Met Ser Ser Thr Asn Ser Ala Leu

305 310 315 320

Leu Leu Ser Ile Leu Lys Met Ile Asp Lys Tyr Asp Leu Tyr Gly Gln

325 330 335

Val Ala Tyr Pro Lys His His Lys Gln Ser Asp Val Pro Asp Ile Tyr

340 345 350

Arg Leu Ala Ala Lys Thr Lys Gly Val Phe Ile Asn Pro Ala Phe Ile

355 360 365

Glu Pro Phe Gly Leu Thr Leu Ile Glu Ala Ala Ala Tyr Gly Leu Pro

370 375 380

Met Val Ala Thr Lys Asn Gly Gly Pro Val Asp Ile His Arg Val Leu

385 390 395 400

Asp Asn Gly Leu Leu Val Asp Pro His Asp Gln Gln Ala Ile Ala Asp

405 410 415

Ala Leu Leu Lys Leu Val Ala Asp Lys Gln Leu Trp Ala Lys Cys Arg

420 425 430

Ala Asn Gly Leu Lys Asn Ile His Leu Phe Ser Trp Pro Glu His Cys

435 440 445

Lys Thr Tyr Leu Ser Arg Ile Ala Ser Cys Lys Pro Arg Gln Pro Arg

450 455 460

Trp Leu Arg Ser Ile Asp Asp Asp Asp Glu Asn Ser Glu Thr Asp Ser

465 470 475 480

Pro Ser Asp Ser Leu Arg Asp Ile His Asp Ile Ser Leu Asn Leu Arg

485 490 495

Phe Ser Leu Asp Gly Glu Lys Asn Asp Asn Lys Glu Asn Ala Asp Asn

500 505 510

Thr Leu Asp Pro Glu Val Arg Arg Ser Lys Leu Glu Asn Ala Val Leu

515 520 525

Ser Leu Ser Lys Gly Ala Leu Lys Ser Thr Ser Lys Ser Trp Ser Ser

530 535 540

Asp Lys Ala Asp Gln Asn Pro Gly Ala Gly Lys Phe Pro Ala Ile Arg

545 550 555 560

Arg Arg Arg His Ile Phe Val Ile Ala Val Asp Cys Asp Ala Ser Ser

565 570 575

Gly Leu Ser Gly Ser Val Lys Lys Ile Phe Glu Ala Val Glu Lys Glu

580 585 590

Arg Ala Glu Gly Ser Ile Gly Phe Ile Leu Ala Thr Ser Phe Asn Ile

595 600 605

Ser Glu Val Gln Ser Phe Leu Leu Ser Glu Gly Met Asn Pro Thr Asp

610 615 620

Phe Asp Ala Tyr Ile Cys Asn Ser Gly Gly Asp Leu Tyr Tyr Ser Ser

625 630 635 640

Phe His Ser Glu Gln Asn Pro Phe Val Val Asp Leu Tyr Tyr His Ser

645 650 655

His Ile Glu Tyr Arg Trp Gly Gly Glu Gly Leu Arg Lys Thr Leu Val

660 665 670

Arg Trp Ala Ala Ser Ile Ile Asp Lys Asn Gly Glu Asn Gly Asp His

675 680 685

Ile Val Val Glu Asp Glu Asp Asn Ser Ala Asp Tyr Cys Tyr Thr Phe

690 695 700

Lys Val Cys Lys Pro Gly Thr Val Pro Pro Ser Lys Glu Leu Arg Lys

705 710 715 720

Val Met Arg Ile Gln Ala Leu Arg Cys His Ala Val Tyr Cys Gln Asn

725 730 735

Gly Ser Arg Ile Asn Val Ile Pro Val Leu Ala Ser Arg Ser Gln Ala

740 745 750

Leu Arg Tyr Leu Tyr Leu Arg Trp Gly Met Asp Leu Ser Lys Leu Val

755 760 765

Val Phe Val Gly Glu Ser Gly Asp Thr Asp Tyr Glu Gly Leu Ile Gly

770 775 780

Gly Leu Arg Lys Ala Val Ile Met Lys Gly Leu Cys Thr Asn Ala Ser

785 790 795 800

Ser Leu Ile His Gly Asn Arg Asn Tyr Pro Leu Ser Asp Val Leu Pro

805 810 815

Phe Asp Ser Pro Asn Val Ile Gln Ala Asp Glu Glu Cys Ser Ser Thr

820 825 830

Glu Ile Arg Cys Leu Leu Val Lys Leu Ala Val Leu Lys Gly

835 840 845

3

atttttttct ctaagttctc tctcgctgtc cttatcattt caccacctcc ataaatctag 60

aaacatcttt tctactccgt taatctctct agcacacggc ggaggagtgc ggcggaggag 120

atg gcg gga aac gat tgg att aac agt tac tta gag gcg ata ctg gat 168

Met Ala Gly Asn Asp Trp Ile Asn Ser Tyr Leu Glu Ala Ile Leu Asp

1 5 10 15

gtt gga cca ggg cta gat gat aag aag tca tcg ttg ttg ttg aga gaa 216

Val Gly Pro Gly Leu Asp Asp Lys Lys Ser Ser Leu Leu Leu Arg Glu

20 25 30

aga ggg agg ttt agt ccg acg agg tac ttt gtt gag gaa gtt att act 264

Arg Gly Arg Phe Ser Pro Thr Arg Tyr Phe Val Glu Glu Val Ile Thr

35 40 45

gga ttc gat gag act gat ttg cat cgt tcg tgg atc cga gca caa gct 312

Gly Phe Asp Glu Thr Asp Leu His Arg Ser Trp Ile Arg Ala Gln Ala

50 55 60

act cgg agt ccg cag aga agg aat act agg ctc gag aat atg tgc tgg 360

Thr Arg Ser Pro Gln Arg Arg Asn Thr Arg Leu Glu Asn Met Cys Trp

65 70 75 80

agg att tgg aat ttg gct cgc cag aaa aag cag ctt gag gga gag caa 408

Arg Ile Trp Asn Leu Ala Arg Gln Lys Lys Gln Leu Glu Gly Glu Gln

85 90 95

gct cag tgg atg gca aaa cgc cgt caa gaa cgt gaa aga ggt cgc aga 456

Ala Gln Trp Met Ala Lys Arg Arg Gln Glu Arg Glu Arg Gly Arg Arg

100 105 110

gaa gca gtt gct gat atg tca gag gat cta tct gag gga gag aaa gga 504

Glu Ala Val Ala Asp Met Ser Glu Asp Leu Ser Glu Gly Glu Lys Gly

115 120 125

gat ata gtc gct gac atg tca tct cat ggt gaa agt acc aga ggc cga 552

Asp Ile Val Ala Asp Met Ser Ser His Gly Glu Ser Thr Arg Gly Arg

130 135 140

ttg cct aga atc agt tct gtt gag aca atg gaa gca tgg gtc agt cag 600

Leu Pro Arg Ile Ser Ser Val Glu Thr Met Glu Ala Trp Val Ser Gln

145 150 155 160

cag aga gga aag aag ctt tat atc gtg ctt ata agt tta cat ggt tta 648

Gln Arg Gly Lys Lys Leu Tyr Ile Val Leu Ile Ser Leu His Gly Leu

165 170 175

att cgg ggt gag aat atg gag ctt gga cgg gat tct gat act ggt ggt 696

Ile Arg Gly Glu Asn Met Glu Leu Gly Arg Asp Ser Asp Thr Gly Gly

180 185 190

cag gtg aag tat gtt gtt gaa ctt gcg agg gcc tta ggg tcg atg cca 744

Gln Val Lys Tyr Val Val Glu Leu Ala Arg Ala Leu Gly Ser Met Pro

195 200 205

ggt gta tat cgg gtt gac ttg ctt act aga caa gta tct tca cca gaa 792

Gly Val Tyr Arg Val Asp Leu Leu Thr Arg Gln Val Ser Ser Pro Glu

210 215 220

gta gat tgg agc tat ggt gag ccg aca gag atg ctg acg cca ata agt 840

Val Asp Trp Ser Tyr Gly Glu Pro Thr Glu Met Leu Thr Pro Ile Ser

225 230 235 240

aca gac ggc ttg atg act gag atg ggg gag agt agt ggt gct tat att 888

Thr Asp Gly Leu Met Thr Glu Met Gly Glu Ser Ser Gly Ala Tyr Ile

245 250 255

att cgc att cct ttt gga cca aga gag aaa tat att cca aaa gaa cag 936

Ile Arg Ile Pro Phe Gly Pro Arg Glu Lys Tyr Ile Pro Lys Glu Gln

260 265 270

cta tgg ccc tat att ccc gaa ttt gtt gat ggt gca ctt aac cat att 984

Leu Trp Pro Tyr Ile Pro Glu Phe Val Asp Gly Ala Leu Asn His Ile

275 280 285

att caa atg tcc aaa gtt ctt ggg gag caa att ggt agt ggc tat cct 1032

Ile Gln Met Ser Lys Val Leu Gly Glu Gln Ile Gly Ser Gly Tyr Pro

290 295 300

gtg tgg cct gtt gcc ata cac gga cat tat gct gat gct ggc gac tca 1080

Val Trp Pro Val Ala Ile His Gly His Tyr Ala Asp Ala Gly Asp Ser

305 310 315 320

gct gct ctc ctg tca ggt gct tta aat gta cca atg ctt ttc act ggt 1128

Ala Ala Leu Leu Ser Gly Ala Leu Asn Val Pro Met Leu Phe Thr Gly

325 330 335

cac tca ctt ggt aga gat aag ttg gag caa ctg ttg gca caa ggt cga 1176

His Ser Leu Gly Arg Asp Lys Leu Glu Gln Leu Leu Ala Gln Gly Arg

340 345 350

aag tca aag gat gaa ata aac tca acc tac aag ata atg cgg aga ata 1224

Lys Ser Lys Asp Glu Ile Asn Ser Thr Tyr Lys Ile Met Arg Arg Ile

355 360 365

gag gct gaa gaa tta act ctt gat gct tcc gaa att gtc atc act agt 1272

Glu Ala Glu Glu Leu Thr Leu Asp Ala Ser Glu Ile Val Ile Thr Ser

370 375 380

aca aga cag gag att gac gag caa tgg cgt ttg tat gat ggg ttt gat 1320

Thr Arg Gln Glu Ile Asp Glu Gln Trp Arg Leu Tyr Asp Gly Phe Asp

385 390 395 400

cca ata tta gag cgt aag tta cgt gca agg atc aag cgc aat gtc agc 1368

Pro Ile Leu Glu Arg Lys Leu Arg Ala Arg Ile Lys Arg Asn Val Ser

405 410 415

tgt tat ggc agg ttt atg cct cgt atg gct gta att cct cct ggg atg 1416

Cys Tyr Gly Arg Phe Met Pro Arg Met Ala Val Ile Pro Pro Gly Met

420 425 430

gag ttc cac cat att gtg cca cat gaa ggt gac atg gat ggt gaa aca 1464

Glu Phe His His Ile Val Pro His Glu Gly Asp Met Asp Gly Glu Thr

435 440 445

gaa gga agt gaa gat ggg aag acc ccg gat cca cct att tgg gca gag 1512

Glu Gly Ser Glu Asp Gly Lys Thr Pro Asp Pro Pro Ile Trp Ala Glu

450 455 460

att atg cgc ttc ttt tct aat cca agg aag cct atg ata ctc gca ctt 1560

Ile Met Arg Phe Phe Ser Asn Pro Arg Lys Pro Met Ile Leu Ala Leu

465 470 475 480

gct agg cct gat ccc aag aag aac ctc act act tta gtg aaa gca ttt 1608

Ala Arg Pro Asp Pro Lys Lys Asn Leu Thr Thr Leu Val Lys Ala Phe

485 490 495

ggt gaa tgt cgt cca ttg aga gag ctt gct aat ctt act ttg ata atg 1656

Gly Glu Cys Arg Pro Leu Arg Glu Leu Ala Asn Leu Thr Leu Ile Met

500 505 510

ggt aat cga gat aat atc gac gaa atg tct agc acc aat tct gca ctt 1704

Gly Asn Arg Asp Asn Ile Asp Glu Met Ser Ser Thr Asn Ser Ala Leu

515 520 525

ctt ctt tca atc ttg aaa atg ata gat aag tat gat ctt tat ggt caa 1752

Leu Leu Ser Ile Leu Lys Met Ile Asp Lys Tyr Asp Leu Tyr Gly Gln

530 535 540

gta gct tat cct aaa cac cac aag cag tca gat gtt cct gat atc tac 1800

Val Ala Tyr Pro Lys His His Lys Gln Ser Asp Val Pro Asp Ile Tyr

545 550 555 560

cgt ctt gct gca aag act aag ggt gtt ttt att aat cca gct ttt att 1848

Arg Leu Ala Ala Lys Thr Lys Gly Val Phe Ile Asn Pro Ala Phe Ile

565 570 575

gag cct ttt gga ctg act ttg att gag gca gca gct tat ggt ctc cca 1896

Glu Pro Phe Gly Leu Thr Leu Ile Glu Ala Ala Ala Tyr Gly Leu Pro

580 585 590

atg gta gcc aca aaa aat gga gga cct gtt gat ata cat agg gtt ctt 1944

Met Val Ala Thr Lys Asn Gly Gly Pro Val Asp Ile His Arg Val Leu

595 600 605

gac aat ggt ctc tta gtg gat ccc cat gat cag cag gca att gct gat 1992

Asp Asn Gly Leu Leu Val Asp Pro His Asp Gln Gln Ala Ile Ala Asp

610 615 620

gct ctt ttg aag ttg gtt gct gat aag caa ctg tgg gct aaa tgc agg 2040

Ala Leu Leu Lys Leu Val Ala Asp Lys Gln Leu Trp Ala Lys Cys Arg

625 630 635 640

gca aat gga tta aaa aat atc cac ctt ttc tca tgg ccc gag cac tgt 2088

Ala Asn Gly Leu Lys Asn Ile His Leu Phe Ser Trp Pro Glu His Cys

645 650 655

aaa act tat cta tcc cgg ata gct agc tgc aaa cca agg caa cca cgc 2136

Lys Thr Tyr Leu Ser Arg Ile Ala Ser Cys Lys Pro Arg Gln Pro Arg

660 665 670

tgg ctg aga tcc att gat gat gat gat gaa aat tca gaa aca gat tca 2184

Trp Leu Arg Ser Ile Asp Asp Asp Asp Glu Asn Ser Glu Thr Asp Ser

675 680 685

cct agt gat tcc ttg aga gat att cat gat ata tct ctg aat ttg aga 2232

Pro Ser Asp Ser Leu Arg Asp Ile His Asp Ile Ser Leu Asn Leu Arg

690 695 700

ttt tca tta gat ggg gaa aag aat gac aat aaa gaa aat gct gat aat 2280

Phe Ser Leu Asp Gly Glu Lys Asn Asp Asn Lys Glu Asn Ala Asp Asn

705 710 715 720

aca tta gac ccc gaa gtt cga agg agc aag tta gag aat gct gtt ttg 2328

Thr Leu Asp Pro Glu Val Arg Arg Ser Lys Leu Glu Asn Ala Val Leu

725 730 735

tcc tta tct aag ggt gca ctg aag agc aca tca aaa tct tgg tcg tca 2376

Ser Leu Ser Lys Gly Ala Leu Lys Ser Thr Ser Lys Ser Trp Ser Ser

740 745 750

gac aag gca gac caa aac cct ggt gct ggt aaa ttc cca gcg att agg 2424

Asp Lys Ala Asp Gln Asn Pro Gly Ala Gly Lys Phe Pro Ala Ile Arg

755 760 765

agg agg cga cat att ttt gtt att gca gtg gat tgt gat gct agc tca 2472

Arg Arg Arg His Ile Phe Val Ile Ala Val Asp Cys Asp Ala Ser Ser

770 775 780

gga ctc tct gga agt gtg aaa aag ata ttt gag gct gta gag aag gaa 2520

Gly Leu Ser Gly Ser Val Lys Lys Ile Phe Glu Ala Val Glu Lys Glu

785 790 795 800

agg gca gag ggt tcc att gga ttt atc ctg gct aca tct ttc aat ata 2568

Arg Ala Glu Gly Ser Ile Gly Phe Ile Leu Ala Thr Ser Phe Asn Ile

805 810 815

tca gaa gta cag tct ttc ctg ctt tca gag ggc atg aat cct act gat 2616

Ser Glu Val Gln Ser Phe Leu Leu Ser Glu Gly Met Asn Pro Thr Asp

820 825 830

ttt gat gct tac ata tgc aat agt ggt ggt gat ctt tat tat tcg tcc 2664

Phe Asp Ala Tyr Ile Cys Asn Ser Gly Gly Asp Leu Tyr Tyr Ser Ser

835 840 845

ttc cat tct gag caa aat cct ttt gta gtt gac ttg tac tat cac tca 2712

Phe His Ser Glu Gln Asn Pro Phe Val Val Asp Leu Tyr Tyr His Ser

850 855 860

cat att gag tat cgt tgg ggg ggc gaa gga ttg aga aag act ttg gtg 2760

His Ile Glu Tyr Arg Trp Gly Gly Glu Gly Leu Arg Lys Thr Leu Val

865 870 875 880

cgt tgg gcc gcc tct atc att gat aag aat ggt gaa aat gga gat cac 2808

Arg Trp Ala Ala Ser Ile Ile Asp Lys Asn Gly Glu Asn Gly Asp His

885 890 895

att gtt gtt gag gat gaa gac aat tca gct gac tac tgc tat act ttc 2856

Ile Val Val Glu Asp Glu Asp Asn Ser Ala Asp Tyr Cys Tyr Thr Phe

900 905 910

aaa gtc tgc aag cct ggg acg gtt cct cca tct aaa gag ctt aga aaa 2904

Lys Val Cys Lys Pro Gly Thr Val Pro Pro Ser Lys Glu Leu Arg Lys

915 920 925

gta atg cga att cag gca ctt cgt tgt cac gct gtt tat tgt caa aat 2952

Val Met Arg Ile Gln Ala Leu Arg Cys His Ala Val Tyr Cys Gln Asn

930 935 940

ggg agt agg att aat gtg atc cct gta ctg gca tct cgg tcc caa gca 3000

Gly Ser Arg Ile Asn Val Ile Pro Val Leu Ala Ser Arg Ser Gln Ala

945 950 955 960

ctc agg tac tta tat ctg cga tgg gga atg gac ttg tcg aag ttg gtg 3048

Leu Arg Tyr Leu Tyr Leu Arg Trp Gly Met Asp Leu Ser Lys Leu Val

965 970 975

gtt ttc gtc gga gaa agt ggt gat acc gat tat gaa gga tta atc ggt 3096

Val Phe Val Gly Glu Ser Gly Asp Thr Asp Tyr Glu Gly Leu Ile Gly

980 985 990

ggt cta cgc aag gct gtc ata atg aaa ggc ctc tgc act aat gca agc 3144

Gly Leu Arg Lys Ala Val Ile Met Lys Gly Leu Cys Thr Asn Ala Ser

995 1000 1005

agc tta att cac ggt aat agg aat tac ccg cta tct gat gtt tta cca 3192

Ser Leu Ile His Gly Asn Arg Asn Tyr Pro Leu Ser Asp Val Leu Pro

1010 1015 1020

ttc gac agc cct aat gtc atc caa gcg gac gag gaa tgt agc agc acc 3240

Phe Asp Ser Pro Asn Val Ile Gln Ala Asp Glu Glu Cys Ser Ser Thr

1025 1030 1035 1040

gaa atc cgt tgc tta ctg gag aaa cta gcg gta ctc aaa gga 3282

Glu Ile Arg Cys Leu Leu Glu Lys Leu Ala Val Leu Lys Gly

1045 1050

taataccctt ccccctttga ttgtcaaaaa cctatatgag ctataagact atgccatgaa 3342

aagaatggcc atccatttgg cttgtctttt gaagctgtta atacttttca acagactaca 3402

aaatgagatg agtcctttga tcctctttaa aggacataaa agctttatgc aagaaccagt 3462

gctgtaaagt tatagaattt cttttgctat atatgacatt cgacagaacc agttccggtt 3522

catcgagaaa aagaaataaa tttcaactta taaacatgcc tgatcatgta aattatcata 3582

tacatccatc ggaaggcatt atcgatgggt tatcagattt ttt 3625

4

1054

Solanum tuberosum

4

Met Ala Gly Asn Asp Trp Ile Asn Ser Tyr Leu Glu Ala Ile Leu Asp

1 5 10 15

Val Gly Pro Gly Leu Asp Asp Lys Lys Ser Ser Leu Leu Leu Arg Glu

20 25 30

Arg Gly Arg Phe Ser Pro Thr Arg Tyr Phe Val Glu Glu Val Ile Thr

35 40 45

Gly Phe Asp Glu Thr Asp Leu His Arg Ser Trp Ile Arg Ala Gln Ala

50 55 60

Thr Arg Ser Pro Gln Arg Arg Asn Thr Arg Leu Glu Asn Met Cys Trp

65 70 75 80

Arg Ile Trp Asn Leu Ala Arg Gln Lys Lys Gln Leu Glu Gly Glu Gln

85 90 95

Ala Gln Trp Met Ala Lys Arg Arg Gln Glu Arg Glu Arg Gly Arg Arg

100 105 110

Glu Ala Val Ala Asp Met Ser Glu Asp Leu Ser Glu Gly Glu Lys Gly

115 120 125

Asp Ile Val Ala Asp Met Ser Ser His Gly Glu Ser Thr Arg Gly Arg

130 135 140

Leu Pro Arg Ile Ser Ser Val Glu Thr Met Glu Ala Trp Val Ser Gln

145 150 155 160

Gln Arg Gly Lys Lys Leu Tyr Ile Val Leu Ile Ser Leu His Gly Leu

165 170 175

Ile Arg Gly Glu Asn Met Glu Leu Gly Arg Asp Ser Asp Thr Gly Gly

180 185 190

Gln Val Lys Tyr Val Val Glu Leu Ala Arg Ala Leu Gly Ser Met Pro

195 200 205

Gly Val Tyr Arg Val Asp Leu Leu Thr Arg Gln Val Ser Ser Pro Glu

210 215 220

Val Asp Trp Ser Tyr Gly Glu Pro Thr Glu Met Leu Thr Pro Ile Ser

225 230 235 240

Thr Asp Gly Leu Met Thr Glu Met Gly Glu Ser Ser Gly Ala Tyr Ile

245 250 255

Ile Arg Ile Pro Phe Gly Pro Arg Glu Lys Tyr Ile Pro Lys Glu Gln

260 265 270

Leu Trp Pro Tyr Ile Pro Glu Phe Val Asp Gly Ala Leu Asn His Ile

275 280 285

Ile Gln Met Ser Lys Val Leu Gly Glu Gln Ile Gly Ser Gly Tyr Pro

290 295 300

Val Trp Pro Val Ala Ile His Gly His Tyr Ala Asp Ala Gly Asp Ser

305 310 315 320

Ala Ala Leu Leu Ser Gly Ala Leu Asn Val Pro Met Leu Phe Thr Gly

325 330 335

His Ser Leu Gly Arg Asp Lys Leu Glu Gln Leu Leu Ala Gln Gly Arg

340 345 350

Lys Ser Lys Asp Glu Ile Asn Ser Thr Tyr Lys Ile Met Arg Arg Ile

355 360 365

Glu Ala Glu Glu Leu Thr Leu Asp Ala Ser Glu Ile Val Ile Thr Ser

370 375 380

Thr Arg Gln Glu Ile Asp Glu Gln Trp Arg Leu Tyr Asp Gly Phe Asp

385 390 395 400

Pro Ile Leu Glu Arg Lys Leu Arg Ala Arg Ile Lys Arg Asn Val Ser

405 410 415

Cys Tyr Gly Arg Phe Met Pro Arg Met Ala Val Ile Pro Pro Gly Met

420 425 430

Glu Phe His His Ile Val Pro His Glu Gly Asp Met Asp Gly Glu Thr

435 440 445

Glu Gly Ser Glu Asp Gly Lys Thr Pro Asp Pro Pro Ile Trp Ala Glu

450 455 460

Ile Met Arg Phe Phe Ser Asn Pro Arg Lys Pro Met Ile Leu Ala Leu

465 470 475 480

Ala Arg Pro Asp Pro Lys Lys Asn Leu Thr Thr Leu Val Lys Ala Phe

485 490 495

Gly Glu Cys Arg Pro Leu Arg Glu Leu Ala Asn Leu Thr Leu Ile Met

500 505 510

Gly Asn Arg Asp Asn Ile Asp Glu Met Ser Ser Thr Asn Ser Ala Leu

515 520 525

Leu Leu Ser Ile Leu Lys Met Ile Asp Lys Tyr Asp Leu Tyr Gly Gln

530 535 540

Val Ala Tyr Pro Lys His His Lys Gln Ser Asp Val Pro Asp Ile Tyr

545 550 555 560

Arg Leu Ala Ala Lys Thr Lys Gly Val Phe Ile Asn Pro Ala Phe Ile

565 570 575

Glu Pro Phe Gly Leu Thr Leu Ile Glu Ala Ala Ala Tyr Gly Leu Pro

580 585 590

Met Val Ala Thr Lys Asn Gly Gly Pro Val Asp Ile His Arg Val Leu

595 600 605

Asp Asn Gly Leu Leu Val Asp Pro His Asp Gln Gln Ala Ile Ala Asp

610 615 620

Ala Leu Leu Lys Leu Val Ala Asp Lys Gln Leu Trp Ala Lys Cys Arg

625 630 635 640

Ala Asn Gly Leu Lys Asn Ile His Leu Phe Ser Trp Pro Glu His Cys

645 650 655

Lys Thr Tyr Leu Ser Arg Ile Ala Ser Cys Lys Pro Arg Gln Pro Arg

660 665 670

Trp Leu Arg Ser Ile Asp Asp Asp Asp Glu Asn Ser Glu Thr Asp Ser

675 680 685

Pro Ser Asp Ser Leu Arg Asp Ile His Asp Ile Ser Leu Asn Leu Arg

690 695 700

Phe Ser Leu Asp Gly Glu Lys Asn Asp Asn Lys Glu Asn Ala Asp Asn

705 710 715 720

Thr Leu Asp Pro Glu Val Arg Arg Ser Lys Leu Glu Asn Ala Val Leu

725 730 735

Ser Leu Ser Lys Gly Ala Leu Lys Ser Thr Ser Lys Ser Trp Ser Ser

740 745 750

Asp Lys Ala Asp Gln Asn Pro Gly Ala Gly Lys Phe Pro Ala Ile Arg

755 760 765

Arg Arg Arg His Ile Phe Val Ile Ala Val Asp Cys Asp Ala Ser Ser

770 775 780

Gly Leu Ser Gly Ser Val Lys Lys Ile Phe Glu Ala Val Glu Lys Glu

785 790 795 800

Arg Ala Glu Gly Ser Ile Gly Phe Ile Leu Ala Thr Ser Phe Asn Ile

805 810 815

Ser Glu Val Gln Ser Phe Leu Leu Ser Glu Gly Met Asn Pro Thr Asp

820 825 830

Phe Asp Ala Tyr Ile Cys Asn Ser Gly Gly Asp Leu Tyr Tyr Ser Ser

835 840 845

Phe His Ser Glu Gln Asn Pro Phe Val Val Asp Leu Tyr Tyr His Ser

850 855 860

His Ile Glu Tyr Arg Trp Gly Gly Glu Gly Leu Arg Lys Thr Leu Val

865 870 875 880

Arg Trp Ala Ala Ser Ile Ile Asp Lys Asn Gly Glu Asn Gly Asp His

885 890 895

Ile Val Val Glu Asp Glu Asp Asn Ser Ala Asp Tyr Cys Tyr Thr Phe

900 905 910

Lys Val Cys Lys Pro Gly Thr Val Pro Pro Ser Lys Glu Leu Arg Lys

915 920 925

Val Met Arg Ile Gln Ala Leu Arg Cys His Ala Val Tyr Cys Gln Asn

930 935 940

Gly Ser Arg Ile Asn Val Ile Pro Val Leu Ala Ser Arg Ser Gln Ala

945 950 955 960

Leu Arg Tyr Leu Tyr Leu Arg Trp Gly Met Asp Leu Ser Lys Leu Val

965 970 975

Val Phe Val Gly Glu Ser Gly Asp Thr Asp Tyr Glu Gly Leu Ile Gly

980 985 990

Gly Leu Arg Lys Ala Val Ile Met Lys Gly Leu Cys Thr Asn Ala Ser

995 1000 1005

Ser Leu Ile His Gly Asn Arg Asn Tyr Pro Leu Ser Asp Val Leu Pro

1010 1015 1020

Phe Asp Ser Pro Asn Val Ile Gln Ala Asp Glu Glu Cys Ser Ser Thr

1025 1030 1035 1040

Glu Ile Arg Cys Leu Leu Glu Lys Leu Ala Val Leu Lys Gly

5

atttttttct ctaaattctc tctcactgtc cttatcattt caccacctcc ataaatctag 60

aaacatcttt tctattccgt taatctctct agcacacggc ggagtgcggc ggaggag 117

atg gcg gga aac gac tgg att aac agt tac tta gag gcg ata ctg gat 165

Met Ala Gly Asn Asp Trp Ile Asn Ser Tyr Leu Glu Ala Ile Leu Asp

1 5 10 15

gta gga cca ggg cta gat gat aag aaa tca tcg ttg ttg ttg aga gaa 213

Val Gly Pro Gly Leu Asp Asp Lys Lys Ser Ser Leu Leu Leu Arg Glu

20 25 30

aga ggg agg ttt agt ccg acg agg tac ttt gtt gag gaa gtt att act 261

Arg Gly Arg Phe Ser Pro Thr Arg Tyr Phe Val Glu Glu Val Ile Thr

35 40 45

gga ttc gat gag act gat ttg cat cgc tcg tgg atc cga gca caa gct 309

Gly Phe Asp Glu Thr Asp Leu His Arg Ser Trp Ile Arg Ala Gln Ala

50 55 60

act cgg agt ccg cag gag agg aat act agg ctc gag aat atg tgc tgg 357

Thr Arg Ser Pro Gln Glu Arg Asn Thr Arg Leu Glu Asn Met Cys Trp

65 70 75 80

agg att tgg aat ttg gct cgc cag aaa aag cag ctt gag gga gag caa 405

Arg Ile Trp Asn Leu Ala Arg Gln Lys Lys Gln Leu Glu Gly Glu Gln

85 90 95

gct cag tgg atg gca aaa cgc cgt caa gaa cgt gag aga ggt cgc aga 453

Ala Gln Trp Met Ala Lys Arg Arg Gln Glu Arg Glu Arg Gly Arg Arg

100 105 110

gaa gca gtt gct gat atg tca gag gat cta tct gag gga gag aaa gga 501

Glu Ala Val Ala Asp Met Ser Glu Asp Leu Ser Glu Gly Glu Lys Gly

115 120 125

gat ata gtc gct gac atg tca tct cat ggt gaa agt acc aga ggc cga 549

Asp Ile Val Ala Asp Met Ser Ser His Gly Glu Ser Thr Arg Gly Arg

130 135 140

ttg cct aga atc agt tct gtt gag aca atg gaa gca tgg gtc agt cag 597

Leu Pro Arg Ile Ser Ser Val Glu Thr Met Glu Ala Trp Val Ser Gln

145 150 155 160

cag aga gga aag aag ctt tat atc gtg ctt ata agt tta cat ggt tta 645

Gln Arg Gly Lys Lys Leu Tyr Ile Val Leu Ile Ser Leu His Gly Leu

165 170 175

att cgg ggt gag aat atg gag ctt gga cgg gat tct gat act ggt ggt 693

Ile Arg Gly Glu Asn Met Glu Leu Gly Arg Asp Ser Asp Thr Gly Gly

180 185 190

cag gtg aag tat gta gtt gga gca act gtt gca caa ggt cgt ttg tca 741

Gln Val Lys Tyr Val Val Gly Ala Thr Val Ala Gln Gly Arg Leu Ser

195 200 205

aag gat gaa ata aac tca acc tac aag ata atg cgg aga ata gag gct 789

Lys Asp Glu Ile Asn Ser Thr Tyr Lys Ile Met Arg Arg Ile Glu Ala

210 215 220

gaa gaa tta act ctt gat gct tcc gaa att gtc atc act agt aca aga 837

Glu Glu Leu Thr Leu Asp Ala Ser Glu Ile Val Ile Thr Ser Thr Arg

225 230 235 240

cag gag att gac gag caa tgg cgt ttg tat gat ggg ttt gat cca ata 885

Gln Glu Ile Asp Glu Gln Trp Arg Leu Tyr Asp Gly Phe Asp Pro Ile

245 250 255

tta gag cgt aag tta cgt gca agg atc aag cgc aat gtc agc tgt tat 933

Leu Glu Arg Lys Leu Arg Ala Arg Ile Lys Arg Asn Val Ser Cys Tyr

260 265 270

ggc agg ttt atg cct cgt atg gct gta att cct cct ggg atg gag ttc 981

Gly Arg Phe Met Pro Arg Met Ala Val Ile Pro Pro Gly Met Glu Phe

275 280 285

cac cat att gtg cca cat gaa ggt gac atg gat ggt gaa aca gaa gga 1029

His His Ile Val Pro His Glu Gly Asp Met Asp Gly Glu Thr Glu Gly

290 295 300

agt gaa gat gga aag acc ccg gat cca cct att tgg gca gag att atg 1077

Ser Glu Asp Gly Lys Thr Pro Asp Pro Pro Ile Trp Ala Glu Ile Met

305 310 315 320

cgc ttc ttt tct aat cca agg aag cct atg ata ctc gca ctt gct agg 1125

Arg Phe Phe Ser Asn Pro Arg Lys Pro Met Ile Leu Ala Leu Ala Arg

325 330 335

cct gat ccc aag aag aac ctc act act tta gtg aaa gca ttt ggt gaa 1173

Pro Asp Pro Lys Lys Asn Leu Thr Thr Leu Val Lys Ala Phe Gly Glu

340 345 350

tgt cgt cca ttg aga gac ctt gct aat ctt act ttg ata atg ggt aat 1221

Cys Arg Pro Leu Arg Asp Leu Ala Asn Leu Thr Leu Ile Met Gly Asn

355 360 365

cga gat aat atc gac gaa atg tct agc acc aat tct gca ctt ctt ctt 1269

Arg Asp Asn Ile Asp Glu Met Ser Ser Thr Asn Ser Ala Leu Leu Leu

370 375 380

tca atc ttg aag atg ata gat aag tat gat ctt tat ggt cta gta gct 1317

Ser Ile Leu Lys Met Ile Asp Lys Tyr Asp Leu Tyr Gly Leu Val Ala

385 390 395 400

tat cct aaa cac cac aag cag tca gat gtt cct gat atc tac cgt ctt 1365

Tyr Pro Lys His His Lys Gln Ser Asp Val Pro Asp Ile Tyr Arg Leu

405 410 415

gct gca aag act aag ggt gtt ttt att aat cca gct ttt att gag cct 1413

Ala Ala Lys Thr Lys Gly Val Phe Ile Asn Pro Ala Phe Ile Glu Pro

420 425 430

ttt gga ctg act ttg att gag gca gca gct tat ggt ctc cca atg gta 1461

Phe Gly Leu Thr Leu Ile Glu Ala Ala Ala Tyr Gly Leu Pro Met Val

435 440 445

gcc aca aaa aat gga gga cct gtt gat ata cat agg gtt ctt gac aat 1509

Ala Thr Lys Asn Gly Gly Pro Val Asp Ile His Arg Val Leu Asp Asn

450 455 460

ggt ctc tta gtg gat ccc cat gat cag cag gca att gct gat gct ctt 1557

Gly Leu Leu Val Asp Pro His Asp Gln Gln Ala Ile Ala Asp Ala Leu

465 470 475 480

ttg aag ttg gtt gct gat aag caa ctg tgg gct aaa tgc agg gca aat 1605

Leu Lys Leu Val Ala Asp Lys Gln Leu Trp Ala Lys Cys Arg Ala Asn

485 490 495

gga tta aaa aat atc cac ctt ttc tca tgg ccc gag cac tgt aaa act 1653

Gly Leu Lys Asn Ile His Leu Phe Ser Trp Pro Glu His Cys Lys Thr

500 505 510

tat cta tcc cgg ata gct agc tgc aaa ccg agg caa cat tcc ttg aga 1701

Tyr Leu Ser Arg Ile Ala Ser Cys Lys Pro Arg Gln His Ser Leu Arg

515 520 525

gat att cat gat ata tct ctg aat ttg aga ttt tca tta gat ggg gaa 1749

Asp Ile His Asp Ile Ser Leu Asn Leu Arg Phe Ser Leu Asp Gly Glu

530 535 540

aag aat gac aat aaa gaa aat gct gat aat aca tta gac ccc gaa gtt 1797

Lys Asn Asp Asn Lys Glu Asn Ala Asp Asn Thr Leu Asp Pro Glu Val

545 550 555 560

cga agg agc aag tta gag aat gct gtt ttg tcc tta tct aag ggt gca 1845

Arg Arg Ser Lys Leu Glu Asn Ala Val Leu Ser Leu Ser Lys Gly Ala

565 570 575

ctg aag agc aca tca aaa tct tgg tcg tca gac aag gca gac caa aat 1893

Leu Lys Ser Thr Ser Lys Ser Trp Ser Ser Asp Lys Ala Asp Gln Asn

580 585 590

cct ggt gct ggt aaa ttc cca gcg att agg agg agg cga cat att ttt 1941

Pro Gly Ala Gly Lys Phe Pro Ala Ile Arg Arg Arg Arg His Ile Phe

595 600 605

gtt att gca gtg gat tgt gat gct agc tca gga ctc tct gga agt atg 1989

Val Ile Ala Val Asp Cys Asp Ala Ser Ser Gly Leu Ser Gly Ser Met

610 615 620

aaa aag ata ttt gag gct gta gag aag gaa agg gca gag ggt tcc att 2037

Lys Lys Ile Phe Glu Ala Val Glu Lys Glu Arg Ala Glu Gly Ser Ile

625 630 635 640

gga ttt atc ctt gct aca tct ttc aat ata tca gaa gta cag tct ttc 2085

Gly Phe Ile Leu Ala Thr Ser Phe Asn Ile Ser Glu Val Gln Ser Phe

645 650 655

ctg ctt tca gag ggc atg aat cct act gag caa aat cct ttt gta gtt 2133

Leu Leu Ser Glu Gly Met Asn Pro Thr Glu Gln Asn Pro Phe Val Val

660 665 670

gac ttg tac tat cac tca cat att gag tat cgt tgg ggg ggc gaa ggg 2181

Asp Leu Tyr Tyr His Ser His Ile Glu Tyr Arg Trp Gly Gly Glu Gly

675 680 685

ttg aga aag act ttg gtg cgt tgg gcc gcc tct atc att gat aag aat 2229

Leu Arg Lys Thr Leu Val Arg Trp Ala Ala Ser Ile Ile Asp Lys Asn

690 695 700

ggt gaa aat gga gat cac att gtt gtt gag gat gaa gac aat tca gct 2277

Gly Glu Asn Gly Asp His Ile Val Val Glu Asp Glu Asp Asn Ser Ala

705 710 715 720

gac tac tgc tat aca ttc aaa gtt tgc aag cct ggg acg gtt cct cca 2325

Asp Tyr Cys Tyr Thr Phe Lys Val Cys Lys Pro Gly Thr Val Pro Pro

725 730 735

tct aaa gaa ctt aga aaa gta atg cga att cag gca ctt cgt tgt cac 2373

Ser Lys Glu Leu Arg Lys Val Met Arg Ile Gln Ala Leu Arg Cys His

740 745 750

gct gtt tat tgt caa aat ggg agt agg att aat gtg atc cct gta ctg 2421

Ala Val Tyr Cys Gln Asn Gly Ser Arg Ile Asn Val Ile Pro Val Leu

755 760 765

gca tct cgg tcc caa gca ctc agg tac tta tat ctg cga tgg gga atg 2469

Ala Ser Arg Ser Gln Ala Leu Arg Tyr Leu Tyr Leu Arg Trp Gly Met

770 775 780

gtc cct gta ctg gca tct cgg tcc caa gca ctc agg tac tta tat ctg 2517

Val Pro Val Leu Ala Ser Arg Ser Gln Ala Leu Arg Tyr Leu Tyr Leu

785 790 795 800

cga tgg gga atg gtc cct gta ctg gca tct cgg tcc caa gca ctc agg 2565

Arg Trp Gly Met Val Pro Val Leu Ala Ser Arg Ser Gln Ala Leu Arg

805 810 815

tac tta tat ctg cga tgg gga atg gac ttg tcg aag ttg gtg gtt ttc 2613

Tyr Leu Tyr Leu Arg Trp Gly Met Asp Leu Ser Lys Leu Val Val Phe

820 825 830

gtc gga gaa agt ggt gat acc gat tat gaa gga ttg atc ggt ggt cta 2661

Val Gly Glu Ser Gly Asp Thr Asp Tyr Glu Gly Leu Ile Gly Gly Leu

835 840 845

cgc aag gct gtc ata atg aaa gga ctc tgc act aat gca agc agc tta 2709

Arg Lys Ala Val Ile Met Lys Gly Leu Cys Thr Asn Ala Ser Ser Leu

850 855 860

att cac ggt aat agg aat tac ccg cta tct gat gtt tta cca ttc gag 2757

Ile His Gly Asn Arg Asn Tyr Pro Leu Ser Asp Val Leu Pro Phe Glu

865 870 875 880

agc cct aat gtc atc caa gcg gat gag gaa tgt agc agc acc gga atc 2805

Ser Pro Asn Val Ile Gln Ala Asp Glu Glu Cys Ser Ser Thr Gly Ile

885 890 895

cgt tcc tta ctg gag aaa cta gcg gta ctc aaa gga taataccctt 2851

Arg Ser Leu Leu Glu Lys Leu Ala Val Leu Lys Gly

900 905

ccccctttga ttgtcaaaaa cctatatgag ctaagattat gccatgaaaa gaatggccat 2911

ccatttggct tgtcttttg 2930

6

908

Solanum tuberosum

6

Met Ala Gly Asn Asp Trp Ile Asn Ser Tyr Leu Glu Ala Ile Leu Asp

1 5 10 15

Val Gly Pro Gly Leu Asp Asp Lys Lys Ser Ser Leu Leu Leu Arg Glu

20 25 30

Arg Gly Arg Phe Ser Pro Thr Arg Tyr Phe Val Glu Glu Val Ile Thr

35 40 45

Gly Phe Asp Glu Thr Asp Leu His Arg Ser Trp Ile Arg Ala Gln Ala

50 55 60

Thr Arg Ser Pro Gln Glu Arg Asn Thr Arg Leu Glu Asn Met Cys Trp

65 70 75 80

Arg Ile Trp Asn Leu Ala Arg Gln Lys Lys Gln Leu Glu Gly Glu Gln

85 90 95

Ala Gln Trp Met Ala Lys Arg Arg Gln Glu Arg Glu Arg Gly Arg Arg

100 105 110

Glu Ala Val Ala Asp Met Ser Glu Asp Leu Ser Glu Gly Glu Lys Gly

115 120 125

Asp Ile Val Ala Asp Met Ser Ser His Gly Glu Ser Thr Arg Gly Arg

130 135 140

Leu Pro Arg Ile Ser Ser Val Glu Thr Met Glu Ala Trp Val Ser Gln

145 150 155 160

Gln Arg Gly Lys Lys Leu Tyr Ile Val Leu Ile Ser Leu His Gly Leu

165 170 175

Ile Arg Gly Glu Asn Met Glu Leu Gly Arg Asp Ser Asp Thr Gly Gly

180 185 190

Gln Val Lys Tyr Val Val Gly Ala Thr Val Ala Gln Gly Arg Leu Ser

195 200 205

Lys Asp Glu Ile Asn Ser Thr Tyr Lys Ile Met Arg Arg Ile Glu Ala

210 215 220

Glu Glu Leu Thr Leu Asp Ala Ser Glu Ile Val Ile Thr Ser Thr Arg

225 230 235 240

Gln Glu Ile Asp Glu Gln Trp Arg Leu Tyr Asp Gly Phe Asp Pro Ile

245 250 255

Leu Glu Arg Lys Leu Arg Ala Arg Ile Lys Arg Asn Val Ser Cys Tyr

260 265 270

Gly Arg Phe Met Pro Arg Met Ala Val Ile Pro Pro Gly Met Glu Phe

275 280 285

His His Ile Val Pro His Glu Gly Asp Met Asp Gly Glu Thr Glu Gly

290 295 300

Ser Glu Asp Gly Lys Thr Pro Asp Pro Pro Ile Trp Ala Glu Ile Met

305 310 315 320

Arg Phe Phe Ser Asn Pro Arg Lys Pro Met Ile Leu Ala Leu Ala Arg

325 330 335

Pro Asp Pro Lys Lys Asn Leu Thr Thr Leu Val Lys Ala Phe Gly Glu

340 345 350

Cys Arg Pro Leu Arg Asp Leu Ala Asn Leu Thr Leu Ile Met Gly Asn

355 360 365

Arg Asp Asn Ile Asp Glu Met Ser Ser Thr Asn Ser Ala Leu Leu Leu

370 375 380

Ser Ile Leu Lys Met Ile Asp Lys Tyr Asp Leu Tyr Gly Leu Val Ala

385 390 395 400

Tyr Pro Lys His His Lys Gln Ser Asp Val Pro Asp Ile Tyr Arg Leu

405 410 415

Ala Ala Lys Thr Lys Gly Val Phe Ile Asn Pro Ala Phe Ile Glu Pro

420 425 430

Phe Gly Leu Thr Leu Ile Glu Ala Ala Ala Tyr Gly Leu Pro Met Val

435 440 445

Ala Thr Lys Asn Gly Gly Pro Val Asp Ile His Arg Val Leu Asp Asn

450 455 460

Gly Leu Leu Val Asp Pro His Asp Gln Gln Ala Ile Ala Asp Ala Leu

465 470 475 480

Leu Lys Leu Val Ala Asp Lys Gln Leu Trp Ala Lys Cys Arg Ala Asn

485 490 495

Gly Leu Lys Asn Ile His Leu Phe Ser Trp Pro Glu His Cys Lys Thr

500 505 510

Tyr Leu Ser Arg Ile Ala Ser Cys Lys Pro Arg Gln His Ser Leu Arg

515 520 525

Asp Ile His Asp Ile Ser Leu Asn Leu Arg Phe Ser Leu Asp Gly Glu

530 535 540

Lys Asn Asp Asn Lys Glu Asn Ala Asp Asn Thr Leu Asp Pro Glu Val

545 550 555 560

Arg Arg Ser Lys Leu Glu Asn Ala Val Leu Ser Leu Ser Lys Gly Ala

565 570 575

Leu Lys Ser Thr Ser Lys Ser Trp Ser Ser Asp Lys Ala Asp Gln Asn

580 585 590

Pro Gly Ala Gly Lys Phe Pro Ala Ile Arg Arg Arg Arg His Ile Phe

595 600 605

Val Ile Ala Val Asp Cys Asp Ala Ser Ser Gly Leu Ser Gly Ser Met

610 615 620

Lys Lys Ile Phe Glu Ala Val Glu Lys Glu Arg Ala Glu Gly Ser Ile

625 630 635 640

Gly Phe Ile Leu Ala Thr Ser Phe Asn Ile Ser Glu Val Gln Ser Phe

645 650 655

Leu Leu Ser Glu Gly Met Asn Pro Thr Glu Gln Asn Pro Phe Val Val

660 665 670

Asp Leu Tyr Tyr His Ser His Ile Glu Tyr Arg Trp Gly Gly Glu Gly

675 680 685

Leu Arg Lys Thr Leu Val Arg Trp Ala Ala Ser Ile Ile Asp Lys Asn

690 695 700

Gly Glu Asn Gly Asp His Ile Val Val Glu Asp Glu Asp Asn Ser Ala

705 710 715 720

Asp Tyr Cys Tyr Thr Phe Lys Val Cys Lys Pro Gly Thr Val Pro Pro

725 730 735

Ser Lys Glu Leu Arg Lys Val Met Arg Ile Gln Ala Leu Arg Cys His

740 745 750

Ala Val Tyr Cys Gln Asn Gly Ser Arg Ile Asn Val Ile Pro Val Leu

755 760 765

Ala Ser Arg Ser Gln Ala Leu Arg Tyr Leu Tyr Leu Arg Trp Gly Met

770 775 780

Val Pro Val Leu Ala Ser Arg Ser Gln Ala Leu Arg Tyr Leu Tyr Leu

785 790 795 800

Arg Trp Gly Met Val Pro Val Leu Ala Ser Arg Ser Gln Ala Leu Arg

805 810 815

Tyr Leu Tyr Leu Arg Trp Gly Met Asp Leu Ser Lys Leu Val Val Phe

820 825 830

Val Gly Glu Ser Gly Asp Thr Asp Tyr Glu Gly Leu Ile Gly Gly Leu

835 840 845

Arg Lys Ala Val Ile Met Lys Gly Leu Cys Thr Asn Ala Ser Ser Leu

850 855 860

Ile His Gly Asn Arg Asn Tyr Pro Leu Ser Asp Val Leu Pro Phe Glu

865 870 875 880

Ser Pro Asn Val Ile Gln Ala Asp Glu Glu Cys Ser Ser Thr Gly Ile

885 890 895

Arg Ser Leu Leu Glu Lys Leu Ala Val Leu Lys Gly

900 905